Modulation of Helix Stability to Investigate Protein-Protein Interactions in the Paramyxovirus Replication Complex

Persistent Link:
http://hdl.handle.net/10150/297509
Title:
Modulation of Helix Stability to Investigate Protein-Protein Interactions in the Paramyxovirus Replication Complex
Author:
Arias, Victor H.
Issue Date:
2013
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
The focus of this project is to develop a greater understanding of the protein-protein interaction between the measles Nucleocapsid Binding Domain (NBD) protein and its ligand N-protein, which are part of the paramyxovirus replication complex. This interaction is an example of a binding reaction where one partner (the N protein) is intrinsically unstructured, but undergoes a coil-to helix transition upon binding. The NBD’s biological role is to bind the N protein which coats the viral RNA genome. However, it must quickly release as the replication complex moves along the RNA during replication. To facilitate this biological function, NBD displays a weak and short-lived interaction with the N-protein that is heavily dependent on the structural stability of both proteins. We are utilizing this pair of proteins as a model system for the study of coupled folding and binding processes. In this work, we have investigated the influence of helix formation in N protein on the NBD-N interaction. Helix stabilizing and destabilizing mutations were introduced into the NBD binding site on the measles N-protein in order to analyze the effects that the structural stability of N-protein have in the binding equilibrium between NBD and N. The measles N protein was mutated by site directed mutagenesis into an unstable mutant, L496G, and a more favorably stable mutant, L496A. These were fused to a small protein, SUMO, to facilitate purification. After, induction and expression of the desired proteins, the proteins were extracted from their respective transformed E. coli BL21-D3 gold expression strain and submitted to protein purification techniques such as Co²⁺ affinity column chromatography, dialysis, and centrifuge concentration. The purity and success of the purification was evaluated by SDS-PAGE electrophoresis, and absorption spectroscopy. The binding reaction between measles NBD wild type, N protein and the mutated variants was analyzed by isothermal titration calorimetry (ITC).
Type:
text; Electronic Thesis
Degree Name:
B.S.
Degree Level:
bachelors
Degree Program:
Honors College; Biology & Biochemistry
Degree Grantor:
University of Arizona
Advisor:
Hausrath, Andrew C.

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleModulation of Helix Stability to Investigate Protein-Protein Interactions in the Paramyxovirus Replication Complexen_US
dc.creatorArias, Victor H.en_US
dc.contributor.authorArias, Victor H.en_US
dc.date.issued2013-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractThe focus of this project is to develop a greater understanding of the protein-protein interaction between the measles Nucleocapsid Binding Domain (NBD) protein and its ligand N-protein, which are part of the paramyxovirus replication complex. This interaction is an example of a binding reaction where one partner (the N protein) is intrinsically unstructured, but undergoes a coil-to helix transition upon binding. The NBD’s biological role is to bind the N protein which coats the viral RNA genome. However, it must quickly release as the replication complex moves along the RNA during replication. To facilitate this biological function, NBD displays a weak and short-lived interaction with the N-protein that is heavily dependent on the structural stability of both proteins. We are utilizing this pair of proteins as a model system for the study of coupled folding and binding processes. In this work, we have investigated the influence of helix formation in N protein on the NBD-N interaction. Helix stabilizing and destabilizing mutations were introduced into the NBD binding site on the measles N-protein in order to analyze the effects that the structural stability of N-protein have in the binding equilibrium between NBD and N. The measles N protein was mutated by site directed mutagenesis into an unstable mutant, L496G, and a more favorably stable mutant, L496A. These were fused to a small protein, SUMO, to facilitate purification. After, induction and expression of the desired proteins, the proteins were extracted from their respective transformed E. coli BL21-D3 gold expression strain and submitted to protein purification techniques such as Co²⁺ affinity column chromatography, dialysis, and centrifuge concentration. The purity and success of the purification was evaluated by SDS-PAGE electrophoresis, and absorption spectroscopy. The binding reaction between measles NBD wild type, N protein and the mutated variants was analyzed by isothermal titration calorimetry (ITC).en_US
dc.typetexten_US
dc.typeElectronic Thesisen_US
thesis.degree.nameB.S.en_US
thesis.degree.levelbachelorsen_US
thesis.degree.disciplineHonors Collegeen_US
thesis.degree.disciplineBiology & Biochemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorHausrath, Andrew C.-
All Items in UA Campus Repository are protected by copyright, with all rights reserved, unless otherwise indicated.